Mechanism of the protective effect of phenylephrine pretreatment against irradiation-induced damage in the submandibular gland

Abstract

Irradiation is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of irradiation treatment is the irreversible damage to salivary glands in the radiation field. Although the protective effect of phenylephrine pretreatment on salivary glands following irradiation has previously been demonstrated, the exact mechanism remains unclear. In this study, we investigated the cytoprotective mechanisms of phenylephrine pretreatment in rat submandibular glands following irradiation. Rats were locally irradiated using a linear accelerator in the head and neck region with a single dose of 20 Gy. Phenylephrine (5 mg/kg) was injected intraperitoneally 30 min prior to irradiation and the submandibular glands were collected on day 7 after irradiation. In comparison with the control group, the irradiation-only group demonstrated severe atrophy, enhanced cell proliferation and increased apoptosis. The phenylephrine-pretreated group, however, demonstrated markedly alleviated atrophy, further increased cell proliferation and decreased apoptosis compared with the irradiation-only group. The data indicated that the cytoprotective mechanisms of phenylephrine pretreatment in the submandibular gland following irradiation may be related to improved cell proliferation and inhibition of cell apoptosis.

Keywords: apoptosis; irradiation; phenylephrine; proliferation; submandibular gland.

Figure 1.

Histopathological alterations of the submandibular gland post-irradiation. All submandibular gland tissues were removed on day 7 post-irradiation. (A) Histopathological structure of the control submandibular gland. (B) The irradiated submandibular gland. (C) The phenylephrine-pretreated submandibular gland. Acinar cells are indicated by (a), ductal cells by (d) and granular convoluted tubule cells by (g). Vacuolization of acinar cells (arrow), pyknotic nuclei (arrowhead) and lysis of entire acini and granular convoluted tubules (empty arrow) were shown in the irradiated submandibular gland and mild atrophy and degeneration were shown in the phenylephrine-pretreated submandibular gland. Sections were stained with hematoxylin and eosin (H&E). Light microscopy magnification is ×400.

Figure 2.

Proliferation in the submandibular gland post-irradiation. Immunohistochemical localizations of PCNA in (A) the control submandibular gland, (B) the irradiated submandibular gland and (C) the phenyl ephrine-pretreated submandibular gland are shown. PCNA-positive cells are indicated by the arrows. Light microscopy magnification is ×400. (D) Semiquantitative scoring of PCNA-positive cells. The PCNA-positive cells were counted in 10 different fields in each section under ×400 magnification. IR, the irradiated submandibular gland; IR + pre-PE, the phenylephrine-pretreated submandibular gland; PCNA, proliferation cell nuclear antigen. ^*P<0.01, compared with the control submandibular gland. ^**P<0.01, compared with the irradiated only submandibular gland.

Figure 3.

Apoptosis in the submandibular gland post-irradiation. The TUNEL-positive cells had a shrunken cell body and condensed nucleus (arrow). (A) In the control submandibular gland, TUNEL staining was rarely observed. (B) In the irradiated submandibular gland, numerous typical densely stained dark TUNEL-positive cell nuclei were observed. (C) In the phenylephrine- pretreated submandibular gland, there were markedly fewer TUNEL-positive cell nuclei than in the irradiated only gland. Light microscopy magnification is ×400. (D) Comparison of the number of TUNEL-positive cells among the different groups. IR, the irradiated submandibular gland; IR+pre-PE, the phenylephrine-pretreated submandibular gland; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. ^*P<0.01, compared with the control submandibular gland. ^**P<0.01, compared with the irradiated only gland.